sign in sign up
<<
Home , Blastocyst, Blastulation, Inner cell mass

Journal für Reproduktionsmedizin und Endokrinologie – Journal of Reproductive Medicine and Endocrinology –

Andrologie • Embryologie & Biologie • Endokrinologie • Ethik & Recht • Genetik Gynäkologie • Kontrazeption • Psychosomatik • Reproduktionsmedizin • Urologie

Morphological Aspects of Human and the Impact of Vitrification Ebner T, Vanderzwalmen P, Shebl O, Mayer RB, Moser M Tews G J. Reproduktionsmed. Endokrinol 2011; 8 (1), 13-20

www.kup.at/repromedizin Online-Datenbank mit Autoren- und Stichwortsuche

Offizielles Organ: AGRBM, BRZ, DVR, DGA, DGGEF, DGRM, D·I·R, EFA, OEGRM, SRBM/DGE

Indexed in EMBASE/Excerpta Medica/Scopus Krause & Pachernegg GmbH, Verlag für Medizin und Wirtschaft, A-3003 Gablitz FERRING-Symposium digitaler DVR 2021 Mission possible – personalisierte Medizin in der Reproduktionsmedizin Was kann die personalisierte Kinderwunschbehandlung in der Praxis leisten? Freuen Sie sich auf eine spannende Diskussion auf Basis aktueller Studiendaten. SAVE THE DATE 02.10.2021 Programm 12.30 – 13.20Uhr Chair: Prof. Dr. med. univ. Georg Griesinger, M.Sc.

12:30 Begrüßung Prof. Dr. med. univ. Georg Griesinger, M.Sc. & Dr. Thomas Leiers

12:35 Sind Sie bereit für die nächste Generation rFSH? Im Gespräch Prof. Dr. med. univ. Georg Griesinger, Dr. med. David S. Sauer, Dr. med. Annette Bachmann

13:05 Die smarte Erfolgsformel: Value Based Healthcare Bianca Koens

13:15 Verleihung Frederik Paulsen Preis 2021

Wir freuen uns auf Sie! Morphology Morphological Aspects of Human Blastocysts and the Impact of Vitrification

T. Ebner1, P. Vanderzwalmen2, O. Shebl1, R. B. Mayer1, M. Moser1, G. Tews1

The topic whether blastocyst culture and transfer is a promising tool in IVF laboratories has been discussed controversially. Discrepancies in outcome may be explained by the fact that formation of a blastocyst on day 5 does not automatically correspond to its viability. Adequate morphological scoring at blastocyst stage (quality of inner mass and trophectoderm, expansion, screening for anomalies) would definitely help to significantly reduce the number of blastocysts being replaced, which in turn would limit the number of multiple gestations. Such a strategy could automatically increase the number of blastocysts to be frozen for later replacement. Currently, vitrification challenges slow freezing as the cryopreservation method of choice, since it is faster, more cost-effective and yields at least comparable thawing results. With respect to this, four morphological parameters of vitrified/warmed blastocyts are reported (re-expansion, hatching, necrotic foci and cytoplasmic defects) which can successfully be used for selection purposes in frozen blastocyst transfer. To conclude, blastocyst culture, transfer and cryopreservation is certainly a valuable method in the hands of IVF practitioners and has gained acceptance by many programs throughout the world. Key words: blastocyst quality, hatching,, re-expansion, vitrification

Morphologische Analyse der Blastozyste und Einfluss der Vitrifikation. Die Meinungen, ob eine Blastozystenkultur bzw. ein Blastozystentransfer vielversprechende Methoden der assistierten Reproduktion sind, gehen auseinander. Ein möglicher Grund für diese Diskrepanzen könnte die Tatsache sein, dass die Bildung einer Blastozyste am 5. Entwicklungstag nicht automatisch bedeutet, dass diese auch vital ist. Eine adäquate morphologische Beurteilung im Blastozystenstadium (Qualität von innerer Zellmasse und Trophektoderm, Expansion, Anomalien) würde sicherlich einer Reduktion der Zahl der zu transferierenden Blastozysten förderlich sein und so weiter zur Verringerung der Mehrlingsschwangerschaften beitragen können. Damit einhergehend würde die Anzahl der zu kryokonservierenden Embryonen natürlich steigen. Um eine exakte Prognose hinsichtlich einer zu erwartenden Implantation nach dem Tauen/Erwärmen erstellen zu können, bieten sich 4 morphologische Parameter zur Selektion ehemals kryokonservierter Blasto- zysten an: Re-Expansion, Hatching, das Vorhandensein nekrotischer Areale sowie zytoplasmatische Defekte. Zusammenfassend lässt sich behaupten, dass bei detaillierter morphologischer Analyse der Blastozyste deren Kultur, Transfer und Kryokonservierung sehr wohl eine Berechtigung auf dem Gebiet der künstlichen Befruchtung hat. J Reproduktionsmed Endokrinol 2011; 8 (1): 13–20. Schlüsselwörter: Blastozystenqualität, Hatching, innere Zellmasse, Re-Expansion, Vitrifikation

„ Introduction delivery, is non-invasive morphological phectoderm (TE) responsible for the ac- selection at different developmental cumulation of fluid in the blastocyst cav- Compared to the natural cycle, the situa- stages [3, 4]. ity and specialised for implantation and tion in IVF is different because con- an inner cell mass (ICM) forming all trolled ovarian hyperstimulation may Theoretically, any prolongation of in three germ layers of the . cause accidental maturation and ovula- vitro culture up to day 4 or day 5 will tion of germ cells of reduced develop- allow for a more accurate prediction of „ Morphology before Vitrifi- mental potential. In other words, the ac- developmental capacity. On day 4 (90– tual implantation potential may be over- 100 hours past insemination), blasto- cation estimated though oocyte morphology, meres should have formed numerous Continual improvement in culture media fertilization and rate may ap- tight intracellular junctions finally re- composition resulting in a higher number pear inconspicuous at first glance. On sulting in a compacting or even a moru- of available day 5 had 2 major the other hand, even embryos of worst la-stage . This marks the switch consequences for embryologists. On the quality may sometimes turn out to be from a cell cluster of individual blas- one hand, adequate cryopreservation viable, e.g. develop to healthy babies. tomeres to a relatively smooth mass with programs for blastocysts had to be estab- indistinguishable cell outlines capable of lished, and, on the other, there was a need However, taken into consideration that actively regulating its internal environ- for more detailed blastocyst scoring sys- usually routine laboratories neither have ment. On the fifth day of in vitro culture tems in order to filter out those blastocysts the equipment nor the resources to ana- (114–120 hours) preimplantation devel- which would implant preferentially. lyze embryo metabolism [1, 2] or cyto- opment should culminate in the forma- genetical constitution, the only approach tion of the blastocyst. Once fully devel- At the beginning of efficient blastocyst to reach the ultimate goal in assisted re- oped human blastocysts consist of two grading some twenty years ago particu- production, namely a healthy singleton different cell types: an outer layer of tro- lar attention was focused on develop-

Received: July 15, 2010; accepted after revision: January 31, 2011 From the 1Landes- Frauen- und Kinderklinik, IVF-Unit, Linz, Austria, and the 2Centre Hospitalier Inter Regional Cavell (CHIREC), Brussels, Belgium and IVF Centers Prof. Zech, Bregenz, Austria Correspondence: Thomas Ebner, PhD, Landes- Frauen- und Kinderklinik, Kinderwunsch Zentrum Linz, A-4020 Linz, Krankenhausstraße 26–30; e-mail: [email protected]

J Reproduktionsmed Endokrinol 2011; 8 (1) 13 For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH. Blastocyst Morphology mental stage, e.g. blastocyst expansion Though it has been suggested [13] that the lineage that gives rise to the fetus [5, 6]. A more recent scoring system [7] indirect assessment of the total cell num- [22]. took additional morphological features ber (TCN) without destroying the blasto- into consideration, namely grade of ex- cyst is possible under good inverted Cell Lineages pansion and morphology of ICM and optics, the vast majority of studies on Hardy et al. [15] were one of the first to TE. According to the degree of expan- TCN were performed using stained cells realize certain interesting differences in sion the blastocysts were scored using of spare embryos of reduced quality (do- the growth rate of both cell lineages. In Roman numbers in ascending order nated to research), thus, probably not general, mitotic rate of the trophecto- ranging from grade I (blastocyst cavity representing the actual cell number of derm is approximately 1.5 times higher less than half of the volume of the em- healthy blastocysts. Early work on TCN than that of the ICM; however, com- bryo) to VI (completely hatched blasto- faced another drawback, namely the in- pared to some other , the over- cyst). Beginning with full blastocyst ability of simple culture media (e.g. all proportion of the inner cell mass is stage (grade III) additional assessment Earle’s balanced salt solution, Ham’s relatively high, e.g. 34% of all cells on of ICM and TE was performed (based on F10, medium T6) to adequately support day 5, 51% on day 6 and 37% on day 7 cell number and cohesion) in order to human embryo development in vitro. [15]. The striking peak on day 6, with predict developmental competence. Apart from achieving lower blastocyst half of all cells in the blastocyst being formation rates [14] these authors some- part of the ICM, is explained by an in- Though the Dokras system [5, 6] was how underestimated mitotic potential of crease in ICM growth rate between days shown to be helpful in routine laboratory in vitro grown blastocysts. 5 and 6, a time when the number of TE work [8] the more detailed Gardner ap- cells is virtually unchanged. However, proach [7] allows for reducing the num- Cell numbers of blastocysts cultured in the next day (day 7) the original ratio is ber of transferred blastocysts without rather simple media ranged from 42.0 ± re-established since TE cells are shown limiting rate [9] and, there- 20.3 to 58.3 ± 8.1 on day 5 [15–18]. to double between days 6 and 7, while fore, gained higher acceptance in IVF However, utilizing sequential media, the the mitotic rate of ICM cells remains laboratories. A recent randomized study rate of was found to be increased, constant [15]. Since there is widespread compared the two scoring systems [10]. e.g. 63.9 ± 5.3 to 110.5 ± 9.9 cells on cell death of even morphologically nor- Although similar numbers of blastocysts day 5 [19], 166.5 ± 16.0 cells on day 6 mal cells in both cell lineages [15] it is were transferred in comparable patient [20] and as many as 284.0 ± 13.5 cells on suspected that the maintenance of cell cohorts, the Gardner score turned out day 7 [20]. The question if co-culture number within the blastocyst cell types to be superior to the Dokras score with feeder cells might positively influ- is regulated by apoptotic phenomena (p < 0.05) in terms of implantation ence TCN [18–20] is discussed contro- [23]. (37.6% vs. 25.0%) and multiple preg- versially [17]. However, it can be sum- nancy (38.6% vs. 17.1%). In addition, marized that a full human blastocyst at „ Inner Cell Mass there was a trend towards higher clinical day 5 of development should exceed pregnancy rates (p = 0.11) with the 60 cells and at least have doubled its cell It can be summarized that the health of a Gardner grading system (66.7% vs. number on day 6. blastocyst is strongly dependent on the 53.0%). overall cell number [24] but also on the It appears quite logical that any phenom- adequate formation of both cell lineages. Blastocyst Cell Number enon that severely reduces cytoplasmic This brings us back to the morphological A factor in common to both scoring sys- volume of the embryo could cause a dra- scoring of inner cell mass and trophecto- tems is the emphasis on blastocyst ex- matic loss of cells at blastocyst stage if derm [7]. According to this system, the pansion on the day of planned transfer. this is reached at all. Indeed published embryoblast is considered to be optimal Developmental stage of the blastocyst reports describe [21] that (grade a) if the ICM showed a tight pack- on days 5 or 6 may range from a retarded loss following cryopreservation resulted age of numerous cells (Fig. 1). Any re- to an expanded or even hatching in significantly lower (p < 0.01) blasto- duction in number and contact affected blastocyst stage and most embryologist cyst cell numbers on day 6 (45.0 ± 3.7) the quality of this cell lineage, thus, rely on visual judgement instead mea- as compared to blastocysts derived from loosely grouped cell accumulations are suring blastocyst diameter. Shapiro et al. fully intact cleavage-stage embryos after scored b whereas absence or presence of [11] accurately measured blastocysts thawing (58.4 ± 3.4). Extensive frag- only few cells randomly distributed prior to transfer and found out that the mentation at earlier stages showed the within the cavity of the blastocyst are diameter of a transferred blastocyst was same detrimental effect on TCN on day 6 classified as grade c (Fig. 2). the most significant variable in predict- [22], e.g. a significant decrease (p < 0.01) ing clinical pregnancy. in cell count from 68.9 ± 5.5 (embryos Optimal blastocysts have been defined without fragmentation) to 29.0 ± 3.6 even more precisely. Quantitative grad- In this context a high variability in cell (> 25% fragmentation). Interestingly, ing of inner cell masses emphasized the numbers has been observed. The mitotic for minimal and moderate levels of frag- importance of ICM size and shape [25]. activity is considered to be a reliable in- mentation the reduction in cell number In contrast to blastocyst expansion and dicator of blastocyst viability and devel- was largely confined to the troph- TE cell number, ICM size was signifi- opmental capacity [12], however, in or- , while a steady number of cantly related to implantation (p < 0.006). der to count the actual number of nuclei ICM cells were maintained. This finding Blastocysts showing an ICM of less than in a blastocyst its cells have to be fixed. suggests a homeostatic regulation of 3800 µm2 showed lower implantation

14 J Reproduktionsmed Endokrinol 2011; 8 (1) Blastocyst Morphology

Figure 2: Blastocyst (IVcb) with no visible inner cell Figure 3: Full blastocyst (IIIbb) showing cell lineages mass (comparable to vesicle). Trophecto- with a limited number of cells. Arrow indicates pres- derm quality is reduced since the left hemisphere con- ence of a necrotic focus in the inner cell mass. sists only of a few large cells. Figure 1: Hatching blastocyst of optimal quality (Vaa) with the hatching site being at the 4 o’clock position. BC: blastocyst cavity; ICM: inner cell mass; TE: trophecto- derm reduction in ICM size of day 6 blasto- with slightly oval (RI: 1.04–1.20) ICMs cysts as compared to day 5 ones (58%). Implantation rates were highest rates (18%) compared to blastocysts (3891 µm2 vs 4458 µm2; p = 0.0016). for embryos with both optimal ICM size with a larger ICM, e.g. > 4500 µm2 and shape (71%). (45%). Present results based on all consecutive single blastocyst transfers of the year Striving to replace blastocyst with large As interesting as these data are, it has 2008 (Tab. 1), however, could only find ICM embryologists should not forget to be emphasized that Richter et al. [25] a significant correlation (p < 0.05) be- that disproportionately oversized ICMs, measured expanded blastocysts of tween size of the embryoblast (5413 vs e.g. with apoptotic processes not work- different size, e.g. ranging from 155– 4141 µm2) and clinical pregnancy at full ing as they are supposed to [22], could 265 µm. Table 1 (unpublished data) in- blastocyst stage (grade III) but not at ex- cause problems in maintaining healthy dicates that the size of the embryoblast is panded stage (grade IV and V). How- central cells (because of the increased closely related to the degree of expan- ever, present data provides first evidence distance over which nutrients and oxy- sion. This seems to be associated with a that size of the embryoblast does not af- gen have to diffuse) and/or could con- more peripheral location of the ICM fect obstetric and neonatal outcome. tribute to large-offspring syndrome [26]. within the blastocyst cavity as the blasto- cyst expands and/or an increased cohe- In addition, the above mentioned authors „ Trophectoderm sion within ICM cells. The latter is fur- [25] evaluated the possible influence of ther supported by the observation that at ICM shape on outcome by introducing In a similar way as ICM, Gardner and full blastocyst stage number of ICM the roundness index (RI) which repre- Schoolcraft [7] classified the TE. The cells can still be estimated (Fig. 3), while sents the length-to-width proportion of outer layer is considered to be optimal if at expanded stage its number can not the inner cell mass. In detail, blastocysts it consists of numerous sickle-shaped be identified accurately. Taking into with extreme RI values of < 1.04 (almost cells forming a cohesive epithelium consideration these empirical data, it round) and > 1.20 (too oval) had a worse (grade a). If number and cohesion of was not surprising that the paper of prognosis (implantation rates of 7% and these cells is somewhat reduced, i.e. Richter and co-workers [25] noticed a 33% respectively) compared to those characterized by the presence of several

Table 1: Obstetric and neonatal outcome of deriving from single blastocyst transfer of blastocysts of different inner cell mass (ICM) size.

Grade III Grade IV Grade V

hCG pos neg pos neg pos neg

n 8 14 22 26 27 28 size of ICM (µm2) 5413 ± 393* 4141 ± 1416* 4275 ± 1264 4579 ± 1630 3898 ± 1421 4271 ± 1710 roundness index (RI) 1.38 ± 0.06 1.29 ± 0.20 1.31 ± 0.24 1.36 ± 0.31 1.38 ± 0.31 1.33 ± 0.33 positive β-hCG 36.4% 45.8% 48.2% clinical pregnancy rate 36.4% 39.6% 41.1% babies born 8 19 24 gestation week 40.5 ± 0.5 39.0 ± 1.0 39.5 ± 1.8 weight 3705 ± 617 3222 ± 525 3250 ± 465 sex ratio (m/f) 1.0 0.39 0.83 malformations 0 1 (5.6) 1 (4.3)

* p < 0.05

J Reproduktionsmed Endokrinol 2011; 8 (1) 15 Blastocyst Morphology

Figure 4: Full blastocyst (IVac) showing rudimentary Figure 5: Inner cell mass of optimal size and shape. Figure 6: Trophoblast vesicle without inner cell mass trophectoderm with necrotic focus (arrow). Reduction in developmental potential is due to the pres- consisting of only a minor number of trophectodermal ence of vacuole (V) and necrotic focus (N). cells. gaps, the TE is scored b. The worst case quality blastocysts only a limited num- better if only TE was affected (32.8%). scenario (grade c) would be a trophecto- ber of papers analyzed the fate of bad Consequently, ICM compactness and derm consisting of very few larger cells quality blastocysts [27, 29]. This group multicellularity contributed more to vi- with a low number of tight junctions of blastocysts with poor prognosis usu- tal implantation than TE cohesiveness (Fig. 4). ally shows lower cell numbers and a [27]. higher degree of chromosomal aberra- Kovacic et al. [27] summarized all tions [29]. Bad quality blastocysts con- Similar to necrotic areas vacuoles at blas- blastocysts showing an impaired tropho- sist of numerous different morphologi- tocyst stage could have a detrimental ef- blast (e.g. extremely flattened cells, no cal subtypes, such as blastocysts with fect on developmental capacity (Fig. 5). sickle-shaped cells, no junctions be- excessive fragmentation, excluded blas- In a recent study, 15.2% (36/237) of day tween TE cells, cells with granulation, tomeres, and necrotic cells (Fig. 5) as 5 blastocysts showed vacuoles [32]. In- pigmentation and/or vacuolization) and well as trophoblast vesicles (Fig. 6). terestingly, a statistically significant noted a 36% implantation rate. This is in trend (p = 0.011) towards an elimination the line of others [11]. These authors The latter type is characterized by the of vacuoles from the inner cell mass counted trophectodermal cells around an absence of the inner cell mass [30] and a could be observed [32] since the vast embryonic equator in one plane of focus rather rudimentary trophectoderm (with majority of vacuoles could be located in and could not find any difference be- only a minor number of nuclei); thus, it the trophectoderm [33] indicating that, tween the mean number of TE cells (per is more or less a trophoblastic vesicle theoretically, embryos can develop strat- plane) and the occurrence of a clinical with a dominant blastocyst cavity that egies to minimize a negative impact of pregnancy. also could be a large vacuole [27]. Only vacuolization on implantation behaviour. 3 out of 26 trophoblast vesicles implanted Reports on pregnancies achieved after Thus, it could be speculated that any pre- (11.5%) after transfer; however, one transfer of vacuolized blastocysts are liminary reduction in trophectodermal abortion reduced live birth rate to 7.7% scarce; however, prognosis was much cell number could easily be compensated [27]. better if vacuolization was restricted to by the accelerated growth rate from day the TE [32]. 6 onwards [15]. The fate of all the other inferior blasto- cyst variations, though they may show Another important characteristic in terms This would be contrary to the data of acceptable ICMs or TEs, will also be of implantation behaviour of blastocysts others [28] who observed that a decrease compromised by larger amounts of frag- is the presence of cytoplasmic extensions in the quality of trophectoderm had an ments or excluded that, on bridging the blastocyst cavity (Fig. 7) at almost linear relationship to a reduced the one hand, will be associated with re- the expanded or later stages (grades IV– rate of live births. In more detail, these duced cell numbers on day 5 [22] and, on VI). These processes are commonly authors reported a 64% live birth rate if the other, may interfere with hatching present in half of the junctional TE cells the trophectoderm was classified as a process per se [31]. Live birth rates (ap- spanning the boundary between the po- and only 13% in the presence of a c-tro- proximately 17%) in these bad quality lar and the mural region of TE and are phectoderm. blastocysts were only marginally in- directed towards the blastocoelic surface creased [27] as compared to trophoblast of the ICM [34]. The cytoplasmic exten- As an illustration it may be emphasized vesicles. sions are thought to be related to the po- that a hatching blastocyst of optimal larized flow of cells from the polar to the quality showing both an ICM and a TE Slightly better results could be achieved mural trophectoderm, consequently they of optimal cohesiveness would be scored when blastocysts showing necrotic foci tend to withdraw as the cells reach their grade Vaa (Fig. 1). in one of the cell lineages had to be final location. Interestingly, variations in transferred. Assessing the actual loca- both shape (from broad triangles to Bad Quality Blastocysts tion of such degenerative areas it turned string-like projections) and length (some Though numerous studies have evalu- out that outcome was worse if ICM was fail to reach the ICM surface) have been ated the outcome of transfer of good affected (23% live births) and slightly observed [34].

16 J Reproduktionsmed Endokrinol 2011; 8 (1) Blastocyst Morphology

Table 2: Implantation and perinatal outcome of transfers with embryos hatching at different spots around the . Modified according to Ebner et al. [38].

haching from hatching from embryonic pole TE

n3284 positive β-hCG 22 (68.8%) 46 (56.8%) clinical PR 22 (68.8%)* 39 (46.4%)* birth 19 (59.4%) 34 (40.5%) IR 27/50 (54.0%)* 52/140 (37.1%)* babies born 20 37 Figure 7: Expanded blastocyst (IVaa) showing charac- gestation week 38.7 ± 2.5 38.2 ± 2.8 teristic cytoplasmic strings (arrows) within the blasto- weight 2976 ± 693 2947 ± 669 coel. sex ratio (m/f) 1.22 1.18 malformations 0 3 (8.1%)

* p < 0.05; IR: implantation rate; n: number of patients; PR: pregnancy rate; TE: trophecto- derm

small vesicles protruding through the Considering the proportions within a zona pellucida. It should be kept in mind blastocyst, the likelihood of blastocysts that this blebbing does not necessarily to hatch from the smaller embryonic site indicate the precise location of subse- is much lower than the chance to herni- quent hatching [33]. However, once a ate near the rather extensive mural tro- small opening has been created the TE phectoderm. As a matter of fact, a recent starts to herniate and – governed by study [38] supports the latter theory Figure 8: Expanded blastocyst (IVaa) that collapsed trophectodermal projections – a larger since out of all hatching blastocysts prior to spontaneous hatching (“breathing”) opening is created by mechanical forces. (Grade V) only 38.9% showed a zona Electron microscopic findings show that breach close to the embryonic pole. Whereas cytoplasmic extensions cover- specialized plump cells, called zona- ing the surface of the inner cell mass are breaker cells [31], line both sides of the Interestingly, Table 2 shows a signifi- a common feature in early blastocysts, trophectoderm at theoretical hatching cantly higher implantation rate if blasto- most of the surface usually is unaffected spots. Superficial microvilli and bundles cysts were transferred that hatched close in expanding blastocysts [34]. Persis- of contractile tonofilaments enable these to the embryoblast (54%) as compared to tence of cytoplasmic strings up to ex- specialists to interact with the ZP, some- blastocysts hatching from the abembry- panded stage possibly marks blastocysts what acting like a sphincter. Additional onic pole (37%). Though, obstetric and of developmental lability. It could indeed mechanical help may come from the neonatal outcome of these single blasto- be an indicator of polarization break- phenomenon of blastocyst “breathing” cyst transfers did not differ, it seems that down, e.g. caused by poor media condi- [33], a sequence of rapid collapses and human blastocysts have a developmental tions, resulting in reduced embryo qual- slow re-expansions considered to assist benefit if they hatch adjacent to the ICM, ity and impaired implantation rates [35]. final extrusion from the ruptured ZP since this area corresponds to the cells (Fig. 8). () that will later Spontaneous Hatching drive invasion into the . Regardless of its respective quality ev- Though the main driving force during Theoretically, hatching close to the ICM ery blastocyst surviving until transfer hatching is a mechanical one, cellular ul- would accelerate contact between those day is forced to escape from its zona pel- trastructure of the zona-breaker cells, es- trophectodermal cells supposed to draw lucida (ZP) in order not to face total ar- pecially the presence of secretory vesicles the blastocyst into the uterine wall and rest of development. such as lysosomes, strongly indicates the endometrium. This mutual inter- that biochemical processes are involved action between blastocyst and While an uterine influence on hatching as well. This is in line with previous may be impaired or delayed if herniation behaviour is likely to exist in vivo [36], work stating that the hatching process takes place opposite the ICM and/or if in vitro spontaneous hatching of the hu- may also be mediated by zona lysins hatching difficulties occur. man embryo is rather supported by the [37]. tremendous increase of internal pressure „ Morphology after Vitrifi- caused by both a gradual accumulation In humans, hatching occurs at various of blastocoelic fluid and cellular prolif- regions of the zona pellucida. While cation eration, mostly of trophectodermal ori- some authors postulate that blastocysts Since more detailed blastocyst scoring gin. show hatching sites mainly close to the systems allow for better prediction of ICM [33], others present contradicting implantation behaviour, steady reduc- In the absence of the uterine milieu the data, finding that most of the blastocysts tion of the number of transferred blasto- hatching process in vitro starts with hatch from the abembryonic pole [31]. cysts is recommended [9]. At the same

J Reproduktionsmed Endokrinol 2011; 8 (1) 17 Blastocyst Morphology time, this strategy increases the number of supernumary blastocysts in culture that have to be stored in liquid nitrogen if the quality of these day 5 concepti is promising in terms of subsequent cryosurvival.

Some 15 years after the successful cryo- preservation of a human blastocyst [39] two major approaches are currently ap- plicable in routine IVF work. Slow ab freezing is a safe and feasible option in human blastocyst cryopreservation and Figure 9: Expanded blastocyst (IVaa) before vitrification (a). Full re-expansion is not reached 2h after warming (b). results in adequate survival and preg- nancy rates [40, 41]. However, since vit- stages compared to full or expanded artefact per se or to laser manipulation rification offers some obvious benefits blastocysts [48–51]. This phenomenon during assisted hatching, though the lat- compared to slow freezing, reports fa- is possibly related to the size of the blas- ter assumption is less probable since voring this rapid freezing technique have tocyst cavity which in turn is correlated zona drilling usually is performed imme- become more frequent in literature [42– to the volume of watery liquid within the diately after warming when the blasto- 44] indicating that for blastocysts it is blastocyst. cyst is shrunk. With respect to this, it is equivalent [45] or even better [46, 47] noteworthy that the rate of complete than slow freezing. Due to the presence and size of the blas- hatching is significantly higher if as- tocyst cavity, vitrified-warmed blasto- sisted hatching is performed near the in- Several factors (unrelated to vitrification cysts experience several morphologic ner cell mass as compared to the oppo- method) are known to directly influence changes and become collapsed during site region [53]. the fate of a cryopreserved blastocyst cryopreservation process. Thus, it is after thawing/warming and transfer. It is more difficult to score a vitrified blasto- One morphological feature of thawed/ important to realize that survival rates in cyst after warming than a fresh one warmed blastocysts that often goes with literature are hardly comparable due to [52]. re-expansion is precocious hatching of the fact that some embryologists focus the blastocyst through the artificial gap on immediate survival while others sug- Re-expansion of the blastocyst cavity created by laser pulses. Quite logically, gest an additional waiting period of 24 (and consequently the blastocyst) after the probability of blastocysts to hatch is hours to facilitate control of growth [43]. thawing/warming is expected within 24 likely to be increased if the conceptus is Differences in implantation rates may be hours [41, 43]. In slow-freezing approxi- already re-expanded. However, even attributed to the fact that not all working mately half of the frozen/thawed blasto- shrunk blastocysts tend to leave their groups apply assisted hatching to the cysts (197/402) turned out to re-expand outer shell if the position within the zona thawed blastocysts (whilst shrunk), immediately after 2–4 hours in culture is close to the opening [38]. Data from though this was found to improve out- [52]. In addition, in vitrified/warmed literature [38] suggest that double clini- come [43]. blastocysts, failure of re-expansion pro- cal pregnancy rates can be achieved cess was found to be associated with (63%) if transferred blastocysts already Most importantly, morphology of the significantly reduced rates of implan- started to hatch compared to blastocysts blastocyst will have a significant impact tation (p = 0.022) and clinical pregnancy without (29%) this morphological at- on survival [43]; therefore, only blasto- (p = 0.021) suggesting fast blastocoelic tribute. When both positive prognostic cysts with good to moderate cell lineages re-expansion of the cavity as a discrimi- markers, re-expansion and hatching, will usually be considered for cryostor- native marker of viability [48]. were combined, every second blasto- age. Nevertheless, cryosurvival of mor- cysts (52.2%) implanted. Blebbing out phologically inconspicuous blastocysts Even if it can be assumed that after sev- of the artificial gap (2 hours after warm- may also fail if they derive from a cohort eral more hours all thawed blastocysts ing) obviously characterizes a subgroup of bad day 3 quality embryos [43]. will re-expand (Fig. 9) any delay in this of blastocysts that can hatch completely process could be the manifestation of without being trapped within the zona A recent publication [48] introduced a altered osmotic and/or metabolic condi- [53]. grading system based on re-expansion, tions. These events are comparable to the hatching (out of the artificial gap in the situation found during blastocoele de- Necrotic Foci and Cytoplasmic zona pellucida), cytoplasmic granulation velopment for the blastocyst cavity Defects and presence of necrotic foci. when water enters the embryo cavity via In contrast to previous positive predic- tight junctions either diffusing passively tors, necrotic foci in warmed blastocysts Re-expansion and Hatching or being pumped actively [33]. It can be are considered to have a negative impact It became evident that the efficiency of hypothesized that in blastocysts with on further development. Like in freezing the vitrification method depends on the delayed re-expansion vitrification may at earlier cleavage stages areas of necro- expansion of the blastocyst, with better have influenced its permeability to sis in thawed blastocysts mark cells that survival in morula or early blastocyst water. This could either be due to a cryo- did not survive vitrification.

18 J Reproduktionsmed Endokrinol 2011; 8 (1) Blastocyst Morphology

There is general agreement that at earlier membranes [57] which should be a small quality of the inner cell mass, quality stages at least 50% of the blastomeres intact line. of the trophectoderm, expansion (e.g. have to survive in order to call a thawed blebbing out of the zona at the embry- embryo viable [21, 54]. Recently, it „ Conclusion onic pole), few anomalies (excluded could be shown that blastomere loss blastomeres, vacuoles, necrotic foci, after thawing in day 2 embryos has a det- Blastocyst culture as the method of cytoplasmic strings). rimental effect on blastocyst formation choice for all patients is a balancing act and cell number [21]. Partially damaged and could result in relative high rates of It should be obligatory to cryopreserve day 3 embryos can be rescued if the ne- cycle cancellation. One prerequisite for supernumary blastocysts of good to crotic blastomeres are removed prior to optimizing blastocyst culture, transfer, moderate quality in order to increase cu- transfer [54, 55]. However, prior to com- and cryopreservation is to develop a bet- mulative pregnancy rate. With regard to paction cell-cell adhesion can be ne- ter understanding of morphological cri- this it is very important to achieve a re- glected and does not reflect cell fusion in teria that may influence the implantation producible outcome, especially in terms blastocysts at all. In other words, re- potential of a certain blastocyst. Numer- of survival after thawing, to allow high moval of necrotic cells is rather impos- ous patient- and oocyte/embryo mor- success rates after frozen/vitrified blas- sible on day 5 and would possibly harm phology related criteria have been sug- tocyst transfer. the affected cell lineages. gested that allow for identification of a certain subgroup of patients who would According to Van den Abbeel et al. [41] Ebner et al. [48] provided first evidence definitely benefit from prolonged culture. the evaluation of immediate blastocyst that partial damage of the blastocyst survival on the basis of morphology is caused by vitrification does not reduce Usually, several blastocysts of different difficult and highly subjective, however, rates of implantation and pregnancy. qualities can be grown per patient which a new scoring system (re-expansion, This is all the more interesting since makes an optimized blastocyst scoring hatching through artificial gap, cytoplas- it played no role whether ICM was system indispensable. With the obvious mic appearance) proved useful in order affected or TE. In detail, of those blasto- current trend towards a significant re- to predict both rates of pregnancy and cysts with known outcome 44% (7/16) duction of transferred embryos, every implantation [48]. implanted if the trophectoderm was mar- effort should be made to filter out the ginally degenerated compared to 37% blastocyst with the highest implantation „ Conflict of Interests (10/27) if minor parts of the ICM potential. With respect to this, it is rec- showed necrosis (p > 0.05). Obviously, ommended to weight the morphological The authors certify, that there is no con- blastocysts can compensate for minor criteria at blastocyst stage as follows: flict of interests in relation to this article. injuries.

This is not the case if the whole cyto- „ Relevancy to Practice plasm is harmed by vitrification. Occa- sionally, blastocyst show extensive To conclude, combining both fresh single blastocyst transfer and single or double granular cytoplasm immediately after frozen blastocyst transfer would not only help to keep the overall hormonal dosage thawing instead of an expected homoge- applied to a patient to a minimum but also to reduce the number of blastocysts per neous appearance. Though these blasto- transfer which would result in a significant reduction of multiple pregnancy rate cysts could be considered for transfer and a higher cumulative pregnancy rate. This would assist to further increase ac- once they have recovered, their implan- ceptance of this method in both patients and clinicians/embryologists. tation potential seems to be limited. „ Relevanz für die Praxis This morphological conspicuousness is different from previous cytoplasmic ir- Ein exaktes Scoring der Blastozyste vor und nach der Kryokonservierung erlaubt regularities such as cytoplasmic pitting, eine gute Prognose hinsichtlich einer Implantation. Dadurch kann die Zahl der zu manifestation of which is probably cul- transferierenden Embryonen weiter reduziert werden, was einem Hauptproblem ture dependent [56]. The granular cyto- der IVF, den Mehrlingsschwangerschaften, entgegenwirkt. plasm sometimes observed after vitrifi- cation is characterized by a halo-like structure in the periphery of the cells (which is most likely to consist of water) References: 5. Dokras A, Sargent IL, Ross C, Gardner RL, Barlow D. The human blastocyst: its morphology and HCG secretion in vitro. 1. Sakkas D, Gardner D. Noninvasive methods to assess em- and a dominant accumulation of some Hum Reprod 1991; 6: 1143–51. cytoskeletal components. Warmed blas- bryo quality. Curr Opin Obstet Gynecol 2005; 17: 283–8. 2. Katz-Jaffe MG and Gardner DK. How proteomics can assist 6. Dokras A, Sargent IL, Barlow DH. Human blastocyst grad- tocysts showing this phenomenon have a in shaping the future of human assisted conception. Reprod ing: an indicator of developmental potential. Hum Reprod reduced capacity to survive vitrification. Biomed Online 2008; 17: 497–501. 1993; 8: 2119–27. In the presence of this cytoplasmic 3. Ebner T, Moser M, Sommergruber M, Tews G. Selection 7. Gardner DK, Schoolcraft WB. In vitro culture of human blas- based on morphological assessment of oocytes and embryos tocysts. In: Jansen R, Mortimer D (eds). Towards Reproductive anomaly, blastocysts with good progno- at different stages of preimplantation development. Hum Certainity: and Genetics Beyond 1999. Parthenon sis for subsequent survival may be dis- Reprod Update 2003; 9: 251–62. Press, Carnforth, 1999; 378–88. 4. Ebner T. Embryo development and assessment of viability. 8. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan tinguished from those with bad progno- In: Gardner DK (ed) In Vitro Fertilization: A Practical Approach. R. Blastocyst quality affects the success of blastocyst-stage sis by healthy appearance of the cell Informa Healthcare, New York, London, 2007; 199–220. . Fertil Steril 2000; 74: 282–7.

J Reproduktionsmed Endokrinol 2011; 8 (1) 19 Blastocyst Morphology

9. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft 27. Kovacic B, Vlaisavljevic V, Reljic M, Cizek-Sajko M. Devel- Zech N. Vitrification of human blastocysts with the Hemi-straw WB. Blastocyst score affects implantation and pregnancy out- opmental capacity of different morphological types of day 5 carrier: application of assisted hatching after thawing. Hum come: towards a single blastocyst transfer. Fertil Steril 2000; human morulae and blastocysts. Reprod Biomed Online 2004; Reprod 2003; 18: 1504–11. 73: 1155–8. 8: 687–94. 44. Vanderzwalmen P, Zech N, Greindl AJ, Ectors F, Lejeune B. 10. Balaban B, Yakin K, Urman B. Randomized comparison of 28. Meintjes M, De Kock A, Smuts M, Basuray R, Chantilis SJ, Cryopreservation of human embryos by vitrification. Gynecol two different blastocyst grading systems. Fertil Steril 2006; Kaufmann RK, Madden JD. The relationship of inner cell Obstet Fertil 2006; 34: 760–9. 85: 559–63. mass-, throphoblast-, blastocyst cavity quality and blastocyst 45. Liebermann J,Tucker MJ. Comparison of vitrification and expansion to live birth. Abstract of the 23rd Annual meeting of 11. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Tho- conventional cryopreservation of day 5 and day 6 blastocysts the ESHRE, 2007. mas S. Large blastocyst diameter, early , and low during clinical application. Fertil Steril 2006; 86: 20–6. preovulatory serum progesterone are dominant predictors of 29. Hardarson T, Caisander G, Sjögren A, Hanson C, Hamberger clinical pregnancy in freah autologous cycles. Fertil Steril L, Lundin K. A morphological and chromosomal study of blasto- 46. Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor 2008; 90: 302–9. cysts developing from morphologically suboptimal human pre- V, Brohammer R, Kato O. Vitrification demonstrates significant embryos compared with control blastocysts. Hum Reprod improvement versus slow freezing of human blastocysts. 12. Brinsko SP, Ball BA, Miller PG, Thomas PG, Ellington JE. In Reprod Biomed Online 2005, 11: 53–7. vitro development of day 2 embryos obtained from young, fer- 2003; 18: 399–407. tile mares and aged, subfertile mares. J Reprod Fertil 1994; 30. Ménézo YJR, Kaufmann R, Veiga A, Servy EJ. A mini-atlas 47. Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. 102: 371–8. of the human blastocyst in vitro. 1999; 7: 61–5. Current aspects of blastocyst cryopreservation. Reprod Biomed Online 2008; 16: 311–20. 13. Bongso A. Defining blastocyst quality. In: Bonso A (ed). 31. Sathananthan H, Menezes J, Gunasheela S. Mechanisms Handbook on Blastocyst Culture. Sydney Press Indusprint, of human blastocyst hatching in vitro. Reprod Biomed Online 48. Ebner T, Vanderzwalmen P, Shebl O, Urdl W, Moser M, Singapore, 1999; 62–79. 2003; 7: 228–34. Zech NH, Tews G. Morphology of vitrified/warmed day-5 em- bryos predicts rates of implantation, pregnancy and live birth. 14. Gardner DK, Lane M. Culture and selection of viable blas- 32. Ebner T, Moser M, Sommergruber M, Gaiswinkler U, Shebl tocysts: a feasible proposition for human IVF? Hum Reprod O, Jesacher K, Tews G. Occurrence and developmental conse- Reprod Biomed Online 2009; 19: 72–8. Update 1997; 3: 367–82. quences of vacuoles throughout preimplantation development. 49. Vanderzwalmen P, Zech H, Prapas Y, Prapas N, Nijs M, 15. Hardy K, Handyside AH, Winston RM. The human blasto- Fertil Steril 2005; 83: 1635–40. Vandamme B, Segal-Bertin G, Debauche C, Lejeune B, van cyst: cell number, death and allocation during late preimplan- 33. Veeck LL, Zaninovic N. Human blastocysts in vitro. In: Roosendaal E, et al. Pregnancy and implantation rates after tation development in vitro. Development 1989; 107: 597–604. Veeck L, Zaninovic N (eds). An Atlas of Human Blastocysts. transfer of fresh and vitrified embryos on day 4 or 5. J Assist Reprod Genet 1999; 16: 147. 16. Winston NJ, Braude PR, Pickering SJ, George MA, Cant A, Parthenon Publishing, Boca Raton, London, New York, Currie J and Johnson MH The incidence of abnormal morphol- Washington, 2003; 99–137. 50. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van ogy and nucleocytoplasmic ratios in 2-, 3- and 5-day human 34. Gardner RL. Flow of cells from polar to mural trophecto- Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, pre-embryos. Hum Reprod 1991; 6: 17–24. derm is polarized in the mouse blastocyst. Hum Reprod 2000; Takahashi K et al. Births after vitrification at morula and 17. Van Blerkom J. Development of human embryos to the 15: 694–701. blastula stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. hatched blastocyst stage in the presence or absence of a 35. Scott LA. Oocyte and embryo polarity. Sem Reprod Med monolayer of Vero cells. Hum Reprod 1993; 8: 1525–39. 2000; 18: 171–83. 51. Zech NH, Lejeune B, Zech H, Vanderzwalmen P. Vitrifica- 18. Vlad M, Walker D and Kennedy RC. Nuclei number in hu- 36. Lin SP, Lee RK, Tsai YJ. In vivo hatching phenomenon of tion of hatching and hatched human blastocysts: effect of an man embryos co-cultered with human ampullary cells. Hum mouse blastocysts during implantation. J Assist Reprod Genet opening in the zona pellucida before vitrification. Reprod Reprod 1996; 11: 1678–86. 2001; 18: 341–5. Biomed Online 2005; 11: 355–61. 19. Dumoulin JCM, Derhaag JG, Bras M, Van Montfoort APA, 37. Schiewe MC, Araujo E, Asch RH, Balmaceda JP. Enzymatic 52. Shu Y, Watt J, Gebhardt J, Dasig J, Appling J, Behr B. The Kester ADM, Evers JLH, Geraedts JPM and Coonen E. Growth characterization of zona pellucida hardening in human eggs value of fast blastocyst re-expansion in the selection of a vi- rate of human preimplantation embryos is sex dependant af- and embryos. J Assist Reprod Genet 1995; 12: 2–7. able thawed blastocyst for transfer. Fertil Steril 2008; 91: ter ICSI but not after IVF. Hum Reprod 2005; 20: 484–91. 401–6. 38. Ebner T, Gruber I, Moser M. Location of herniation pre- 20. Fong CY, Bongso A. Comparison of human blastulation dicts implantation behaviour of hatching blastocysts. J Turkish 53. Miyata H, Matsubayashi H, Fukutomi N, Matsuba J, rates and total cell number in sequential culture media with German Gynecol Assoc 2007; 8: 184–8. Koizumi A, Tomiyama T. Relevance of the site of assisted and without co-culture. Hum Reprod 1998; 14: 774–81. hatching in thawed human blastocysts: a preliminary report. 39. Cohen J, Simons R, Fehilly C, Fishel S, Edwards R, Hewitt Fertil Steril 2010; 94: 2444–7. 21. Archer J, Gook DA, Edgar DH. Blastocyst formation and J, Rowland G, Steptoe P, Webster J. Birth after replacement cell numbers in human frozen-thawed embryos following ex- of hatching blastocyst cryopreserved at expanded blastocyst 54. Rienzi L, Nagy ZP, Ubaldi F, Iacobelli M, Anniballo R, tended culture. Hum Reprod 2003; 18: 1669–73. stage. Lancet 1985; 1: 647. Tesarik J, Greco E. Laser-assisted removal of necrotic blas- 22. Hardy K, Stark J, Winston RML. Maintainance of the inner 40. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Chang- tomeres from cryopreserved embryos that were partially dam- cell mass in human blastocysts from fragmented embryos. ing the start temperature and cooling rate in a slow-freezing aged. Fertil Steril 2002; 77: 1196–201. Biol Reprod 2003; 68: 1165–9. protocol increases human blastocyst viability. Fertil Steril 55. Rienzi L, Ubaldi F, Iacobelli M, Minasi MG, Romano S, 23. Hardy K, Stark J. Mathematical models of the balance be- 2003, 79: 407–10. Ferrero S, Sapienza F, Baroni E, Tesarik J, Greco E. Develop- tween apoptosis and proliferation. Apoptosis 2002; 7: 373–81. 41. Van den Abbeel E, Camus M, Verheyen G, Van Waesberghe mental potential of fully intact and partially damaged cryo- 24. Matsuura K, Hayashi N, Takiue C, Hirata R, Naruse K. L, Devroey P; Van Steirteghem A. Slow controlled-rate freez- preserved embryos after laser-assited removal of necrotic Blastocyst quality scoring based on morphologically grading ing of sequentially cultured human blastocysts: an evaluation blastomeres and post-thaw culture selection. Fertil Steril correlates with cell number. Fertil Steril 2010; 94: 1135–7. of two freezing strategies. Hum Reprod 2005; 20: 2929–45. 2005; 84: 888–94. 25. Richter KS, Harris DC, Daneshmand ST, Shapiro BS. Quan- 42. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, 56. Ebner T, Tews G, Sommergruber M, Moser M. Cytoplasmic titative grading of human blastocyst: optimal inner cell mass Kasai M; Takahashi K. Vitrification of human blastocysts using pitting has a negative influence on implantation outcome. size and shape. Fertil Steril 2001; 76: 1157–67. cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003; J Assist Reprod Genet 2005; 22: 239–44. 26. Leese HJ, Donnay I and Thompson JG. Human assisted 18: 384–91. 57. Zhu SE, Zeng SM, Yu WL, Li SJ, Zhang ZC, Chen YF. Vitrifi- conception: a cautionary tale. Lessons from domestic . 43. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, cation of in vivo and in vitro produced bovine blastocysts. Hum Reprod 1998; 13 (Suppl 4): 184–202. Bollen N, van Roosendaal E, Vandervorst M, Schoysman R and Anim Biotechnol 2001; 12: 193–203.

20 J Reproduktionsmed Endokrinol 2011; 8 (1) Mitteilungen aus der Redaktion

Besuchen Sie unsere Rubrik  Medizintechnik-Produkte

Artis pheno Siemens Healthcare Diagnostics GmbH Neues CRT-D Implantat Philips Azurion: Intica 7 HF-T QP von Biotronik Innovative Bildgebungslösung

Aspirator 3 Labotect GmbH

InControl 1050 Labotect GmbH

e-Journal-Abo Beziehen Sie die elektronischen Ausgaben dieser Zeitschrift hier. Die Lieferung umfasst 4–5 Ausgaben pro Jahr zzgl. allfälliger Sonderhefte. Unsere e-Journale stehen als PDF-Datei zur Verfügung und sind auf den meisten der markt­ üblichen e-Book-Readern, Tablets sowie auf iPad funktionsfähig.  Bestellung e-Journal-Abo

Haftungsausschluss Die in unseren Webseiten publizierten Informationen richten sich ausschließlich an geprüfte­ und autorisierte medizinische Berufsgruppen und entbinden nicht von der ärztlichen Sorg- faltspflicht sowie von einer ausführlichen Patientenaufklärung über therapeutische Optionen und deren Wirkungen bzw. Nebenwirkungen. Die entsprechenden Angaben werden von den Autoren mit der größten Sorgfalt recherchiert und zusammengestellt. Die angegebenen Do- sierungen sind im Einzelfall anhand der Fachinformationen zu überprüfen. Weder die Autoren,­ noch diedie tragendentragenden GesellschaftenGesellschaften nochnoch der der Verlag Verlag übernehmen übernehmen irgendwelche irgendwelche Haftungsan Haftungs-­ ansprüche.sprüche. Bitte beachten Sie auch diese Seiten: Impressum Disclaimers & Copyright Datenschutzerklärung